Bacterial community and cyanotoxin gene distribution of the Winam Gulf, Lake Victoria, Kenya

Abstract The Winam Gulf (Kenya) is frequently impaired by cyanobacterial harmful algal blooms (cHABs) due to inadequate wastewater treatment and excess agricultural nutrient input. While phytoplankton in Lake Victoria have been characterized using morphological criteria, our aim is to identify potential toxin‐producing cyanobacteria using molecular approaches. The Gulf was sampled over two successive summer seasons, and 16S and 18S ribosomal RNA gene sequencing was performed. Additionally, key genes involved in production of cyanotoxins were examined by quantitative PCR. Bacterial communities were spatially variable, forming distinct clusters in line with regions of the Gulf. Taxa associated with diazotrophy were dominant near Homa Bay. On the eastern side, samples exhibited elevated cyrA abundances, indicating genetic capability of cylindrospermopsin synthesis. Indeed, near the Nyando River mouth in 2022, cyrA exceeded 10 million copies L−1 where there were more than 6000 Cylindrospermopsis spp. cells mL−1. In contrast, the southwestern region had elevated mcyE gene (microcystin synthesis) detections near Homa Bay where Microcystis and Dolichospermum spp. were observed. These findings show that within a relatively small embayment, composition and toxin synthesis potential of cHABs can vary dramatically. This underscores the need for multifaceted management approaches and frequent cyanotoxin monitoring to reduce human health impacts.


INTRODUCTION
Cyanobacterial harmful algal blooms (cHABs) are a severe and growing threat to freshwater systems, affecting environmental and human health, fisheries, and recreation.Although considerable attention is paid to cHAB occurrence in North America (including the Laurentian Great Lakes) and Europe, their occurrence and impacts in the global south remain less well characterized.Specifically, Lake Victoria, which is shared by Kenya, Uganda, and Tanzania, is highly eutrophic and has in recent years experienced large toxic blooms of Microcystis spp.along with other cyanobacteria (Frank et al., 2023;Okello & Kurmayer, 2011;Olokotum et al., 2020Olokotum et al., , 2021Olokotum et al., , 2022)).Cyanobacterial blooms are a lake-wide issue but are especially prevalent in the bays and gulfs of Lake Victoria.
Lake Victoria is the second-largest freshwater lake by surface area, and its basin has one of the highest population densities in Africa (Olokotum et al., 2020).Lake Victoria has several large and growing cities on its shores and the catchment is dominated primarily by cropland.Because of the rapid increases in population and agriculture in the area around Lake Victoria, eutrophication is an issue in the lake.Anthropogenic sources of excess nutrients are primarily from rivers that drain heavily farmed watersheds, sewage, and aquaculture operations, while atmospheric deposition of nitrogen (N) and phosphorus (P) from windblown burnt biomass is an additional contributor (Cheruiyot & Muhandiki, 2014;Olokotum et al., 2020).Nutrient dynamics in Lake Victoria have drastically changed as a result of land use change, and more recently, the reopening of the Mbita Channel (Njagi et al., 2022;Simiyu et al., 2022).Diatom communities have shifted to Nitzschia while cyanobacteria have proliferated over the past 50 years (Njagi et al., 2022).Urban area, farmland, and wetlands have increased while woodlands, forests, and grasslands decreased.As a result, total organic carbon, total nitrogen (TN), and P levels have increased in the lake, both in the water column and in sediments (Njagi et al., 2022).
Kenya's third largest city, Kisumu, is situated on the shore of Winam Gulf, and the city's drinking water comes from the Gulf.Along with drinking water for the city, nearshore fishing is a major economic industry in the region, as well as a food source for local subsistence fishers (Roegner et al., 2020).The lake also serves as a water source for drinking, laundry, and other household uses in many villages around the Gulf, where residents reported to use the water even if there was a thick visible green surface scum, many of which were producing microcystins (Obuya et al., 2023;Secaira Ziegler et al., 2022).Government officials in the region often campaign for practices to reduce exposure to toxins and eutrophication of the Gulf.These include treating or filtering water before drinking, avoiding bathing in the lake, and avoiding bringing livestock into the lake.Though these strategies would reduce risk of exposure to cyanotoxins, a lack of enforcement and infrastructure make implementation difficult in many communities (Secaira Ziegler et al., 2022).Children and other sensitive populations in these areas are especially susceptible to exposure to cHAB toxins.The region of Kenya around the Winam Gulf has high rates of HIV, malaria, and schistosomiasis; these diseases affect the immune system and lower body weight, increasing risk associated with longterm exposure to microcystins (Roegner et al., 2020;Secaira Ziegler et al., 2022).
The Winam Gulf in Eastern Lake Victoria is shallow, with an average depth of 4.6 m (Gikuma-Njuru et al., 2013).In a previous study that aimed to characterize phytoplankton community composition, it was found that the Gulf was dominated by Microcystis spp., with diazotrophic Anabaena present when N was limited later in the year (Sitoki et al., 2012).Both genera are microcystin producers, and this same study found microcystins above the WHO contact advisory standard, exceeding 100 μg L À1 (Codd et al., 2005;Sitoki et al., 2012).In the Gulf, P concentrations are lower than those observed in the open lake.Conversely, TN concentrations are lower in the open lake than Gulf due to active denitrification (Gikuma-Njuru & Hecky, 2005).It is believed that soluble reactive phosphorus and total phosphorus (TP) from the open lake are contributing to the total P load in the Gulf (Gikuma-Njuru & Hecky, 2005).Despite there being abundant sources of N from river outflow, TN:TP ratios in the Gulf are lower than the Redfield ratio, suggesting that the conditions are favourable for diazotrophic cyanobacteria (Gikuma-Njuru & Hecky, 2005).
Although a cHAB may appear to be dominated by one or a few key cyanobacteria, there is potential for a variety of genotypes present within the community.Some genotypes of cyanobacteria that are of interest for monitoring efforts are those containing genes from the mcy, sxt, cyr, and atx operons encoding for production of microcystins, saxitoxins, cylindrospermopsins, and anatoxins, respectively (Cullen et al., 2019;Pearson & Neilan, 2008).Toxigenicity is not apparent when viewing the community through a microscope, but if the cyanobacteria contain the biosynthetic gene clusters there is potential for toxin production.Indeed, the environmental triggers for cyanotoxin production are largely unknown and differ for each cyanobacterial species (Paerl & Otten, 2013).This means that analysing the community for toxin-production genes provides a basis for identifying risks and improving monitoring strategies to reduce exposure.
To our knowledge, there have been few studies characterizing water column bacterial communities through 16S rRNA gene amplicon sequencing over the entire Winam Gulf, particularly at regular limnological monitoring sites.Primarily, characterization of cyanobacteria has been done through morphological classifications (Lung'Ayia et al., 2000;Simiyu & Kurmayer, 2022;Sitoki et al., 2012).There are limited molecular studies that have been done in Lake Victoria or the Winam Gulf.Of the studies available, only one aimed to characterize bacterial communities in river outflows and wastewater treatment plant effluents (Wachira et al., 2022).Additionally, one dissertation aimed to characterize cyanobacteria and mcyE through denaturing gradient gel electrophoresis, and one study had a goal of characterizing Microcystis aeruginosa across East Africa using molecular techniques (Chhun, 2007;Haande et al., 2007).Other molecular studies looked at specific bacteria, such as Escherichia coli, Vibrio cholerae, and Salmonella, and this work generally centred around detections of these bacteria in fish (Awuor et al., 2011;Baniga et al., 2020;Hounmanou et al., 2019Hounmanou et al., , 2022)).
The aim of this work is to characterize bacterial and phytoplankton assemblages in the Winam Gulf through 16S and 18S rRNA gene sequencing.Through this, we can examine potentially toxin-forming cyanobacteria in the Gulf and assess potential public health risks to the surrounding communities.A secondary goal of this work is to identify the potential of cyanotoxin production through quantitative polymerase chain reaction (qPCR).By sequencing the community, we can determine if there are regions of the Winam Gulf with a distinct bacterial and/or phytoplankton community structure.Additionally, we can find the toxin production potential of the cHAB and identify what species may be responsible for production of microcystins.Our hypothesis is that sites with distinct spatial characteristics (river inflow, nearshore, offshore) will have more similar bacterial communities to sites of the same type.However, we found that communities clustered by location in the Gulf rather than by landscape or hydrological features.We also found that communities were not dominated by Microcystis spp.and microcystins were generally in low to undetected concentrations, with exceptions to two sites.Furthermore, we identified evidence for a previously undetected cyanotoxin, cylindrospermopsin, that warranted additional sampling the following year.

EXPERIMENTAL PROCEDURES
The study was conducted in the Winam Gulf, a shallow bay along the eastern shore of Lake Victoria.The Gulf is located entirely within Kenya, with a surface area of 1333 km 2 (Gikuma-Njuru et al., 2013).During the period of 22-25 June 2022 for cruise A, 18 sites were sampled off of R.V. Uvumbuzi throughout the Gulf (Figure 1A, B) The 2022 cruise B sampling occurred at 23 sites over 27-30 June 2022.All sites in cruise A, other than Soklo and Asembo Bay, were sampled during both cruises.In 2023, sampling occurred during the period of 29 May-3 June.For the purpose of this study, we define eastern bay sites as Sites 1-7 and 14-18, and western bay sites as Sites 8-13.
At each site, water was collected at 1 m depth three times using a 2.5 L Van Dorn sampler and mixed.A total of 1 m was chosen as the sampling depth as it was shallow enough for no sediment interference and minimal surface scums were reflective of a mixed water column.Microbial biomass was collected during cruise A by filtering water through Sterivex cartridge filter units (Sigma Aldrich, St. Louis, MO) with 0.22 μm pore size.
Samples were preserved by filling cartridges with RNAlater solution (Invitrogen, Thermo Fisher Scientific Inc, Waltham, MA) and frozen at À20 C. At each site, an aliquot of lake water was also preserved with Lugol's iodine solution for identification of cyanobacteria and phytoplankton by microscopy.Physicochemical parameters (i.e., water temperature, dissolved oxygen, conductivity, total dissolved solids, pH) were measured using a multiparameter sonde (YSI Inc., Yellow Springs, OH).
Prior to DNA extraction, RNALater solution was flushed out of the Sterivex filter units with DNase-free water (Invitrogen UltraPure DNase/RNase-Free Distilled Water).Filter cartridges were opened using a pipe cutter and filter membrane was carefully removed.DNA from the filters was extracted using a DNeasy Power-Water kit (Qiagen, Germantown, MD) according to the manufacturer's protocol, and purity and quantity were assessed with a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc.).Extracted DNA was kept frozen at À80 C until downstream analyses.
Quantification of cyanobacterial toxin gene abundances were assessed by quantitative PCR (qPCR) for cruise A using the multiplexed CyanoDTec total cyanobacteria and cyanotoxin gene kits (Phytoxigene, Akron, OH) and a Q-qPCR thermocycler (Quantabio, Beverly, MA).Cycling conditions were selected according to Phytoxigene kit specifications.The total cyanobacteria kit amplifies the 16S rRNA gene of cyanobacteria using a universal primer set and contains an internal amplification control.The multiplexed cyanotoxin kit amplifies key genes involved in synthesis of microcystins/nodularins, saxitoxins, and cylindrospermopsins (mcyE/ ndaF, sxtA, and cyrA, respectively) (Al-Tebrineh et al., 2012).
Sequencing of 16S and 18S ribosomal RNA libraries for cruise A samples was performed at the University of Minnesota Genomics Center using a MiSeq sequencer (2 Â 300 paired-end reads; Illumina, San Diego, CA) and FastQC.Primer set 515F/809R was used to target the V4 region of the 16S rRNA gene for amplification (Gohl et al., 2016).The resulting FASTQ files were analysed in RStudio (version 2022.07.2) using a DADA2-based workflow (version 1.22.0;Callahan et al., 2016).Sequences were trimmed to remove primers and low-quality read ends, filtered (minimum quality score of 28), adjusted in accordance with the DADA2 error correction model, and paired-end reads were merged (minimum overlap of 20 bases).From the merged paired-end reads, an amplicon sequence variant (ASV) table was constructed and chimeric ASVs were removed.Taxonomy was assigned to ASVs using the SILVA 16S reference database (version 132) (Quast et al., 2012).The phyloseq package (v1.38.0) was used to generate relative abundance bar plots and hierarchical clustering analysis using Bray-Curtis dissimilarity (McMurdie & Holmes, 2013).Tidyverse (version 1.3.2) was also used for data visualization (Wickham et al., 2019).Spearman's rank was utilized for correlation analyses and forward selection analyses were performed for the microbial community, microcystins, and mcyE copies.One site, Kisumu Pier, was removed from 16S community analysis due to low read counts and quality scores.The 18S rRNA gene was amplified with primer set 1391F/EukBr targeting the V9 region (Amaral-Zettler et al., 2009).The reads were also processed with the DADA2 workflow without merging paired-end reads, and the Protist Ribosomal Reference database (version 4.14.1) was used for assigning taxonomy to the forward reads (Guillou et al., 2012).
Total microcystin concentration was measured in accordance with USEPA Method 546 for both cruises (U.S. EPA, n.d.).Briefly, whole water was collected at each of the 18 sites and then lysed using three cycles of freeze/thaw.The lysate was then assayed for microcystins using the Microcystins/Nodularins (ADDA) ELISA kit (Gold Standard Diagnostics, Warminster, PA) according to kit instructions.Absorbance was read at 450 nm using a Multiskan FC Microplate Photometer (Thermo Fisher Scientific Inc.).In 2023, cylindrospermopsins were measured using a Cylindrospermopsin ELISA kit (Gold Standard Diagnostics) in addition to microcystins.
To measure chlorophyll-a for both cruise tracks, water was filtered onto 25 mm glass fibre filters (GF/F; Whatman, Maidstone, UK) in varying volumes depending on biomass concentrations and turbidity and placed into a 15 mL polypropylene centrifuge tube.Sample filters were frozen until processing when 5 mL of 90% acetone was added to the tube.Each sample was sonicated for 1 min before storage at 4 C in the dark overnight.Chlorophyll concentrations (μg L À1 ) were calculated by measuring absorbance (750, 665, 645, 630 nm) following a modified EPA Method 446 protocol with adjusted constants using a GENESYS 10 UV-Vis Spectrophotometer (Thermo Fisher Scientific Inc.) and adjusting to volume filtered (Arar, 1997).
Whole water was analysed by microscopy for phytoplankton abundance in a measured aliquot using the Utermöhl method with a magnification modification of the stratified counting technique of Munawar and Munawar (1976)

Water quality variables
Water quality measurements were taken at each of the sites throughout the coastal areas of the Winam Gulf (Figure 2).Water temperature remained stable for sites on the cruise transect with a mean of 25.8 C, fluctuating four degrees Celsius during both cruises.Conductivity was consistent (143.1 ± 14.8 μS cm À2 ), besides two sites with the minimum (108.4μS cm À2 , cruise A) at Mbita East and maximum (179.0 μS cm À2 , cruise B) at the Nyando R. mouth.In contrast, dissolved oxygen (DO; 7.68 ± 2.07 mg L À1 ) showed higher variability, with high values at Maboko Island (12.77 mg L À1 , cruise B) and Oluch (11.46 mg L À1 , cruise A) and low values at Kisumu Pier (4.09 mg L À1 , cruise B) and Dunga (4.86 mg L À1 , cruise B).

Chlorophyll-a and microcystin concentrations
Extracted chlorophyll-a concentrations were measured as a proxy for phytoplankton biomass and were observed in moderate to high concentrations at sampling sites in the Gulf (Table 1).Near the Nyando R. mouth, chlorophyll-a was in lower concentrations than in the embayments and offshore sites (11.69 ± 6.86 μg L À1 vs. 50.37± 79.29 μg L À1 , respectively; p<0.05).
Microcystin concentrations were analysed at all sites but were only detectable (≥0.15 μg L À1 ) at three sites: Mirunda Bay (0.15 μg L À1 ), Achieng Oneko (0.15 μg L À1 ), and Homa Bay (0.19 μg L À1 ) during cruise A. Despite the short time between cruises, microcystin analysis for cruise B revealed concentrations of 2.22 and 1.32 μg L À1 at Homa Bay and Mirunda Bay.Additionally, water taken from a pier along the shore of Homa Bay, as well as water from adjacent to the Homa Bay drinking water plant intake was assayed for microcystins.The intake was below the limit of detection, but the nearshore pier had the highest measured concentration of the entire cruise A survey with a concentration of 1.3 μg L À1 .No apparent correlation between microcystins and chlorophyll-a was observed (r s = 0.15, p = 0.55).In contrast, analysis of cyanotoxins in 2023 revealed low but consistent concentrations of microcystins in the Gulf (0.3 ± 0.1 μg L À1 ), except for samples from the shore of Homa Bay and Kendu Bay where microcystins exceeded 5 μg L À1 .Cylindrospermopsins were found in nonzero concentrations at Kisumu Pier, Dunga, and the Nyando R. mouth (estimated at 0.011, 0.035, and 0.003 μg L À1 , respectively), but were outside the dynamic range of the assay for accurate quantification.

Spatial patterns in bacterioplankton and chloroplast community composition
Overall, the communities resembled those of other eutrophic freshwater lakes, with high relative abundances of Cyanobacteria, Bacteroidetes, Planctomycetes, Proteobacteria, Actinobacteria, and Verrucomicrobia (Figures 3 and S1;Gohl et al., 2016;Callahan et al., 2016;Quast et al., 2012;McMurdie & Holmes, 2013).Less abundant phyla Acidobacteria, Chloroflexi, and Latescibacteria were variable across the Gulf, not being present at all sites and never exceeding 5% at any one site.Dunga and Bala Rawi prokaryotic communities had the highest Shannon's diversity, while Naya had the lowest (S2a).The forward selection analysis on the microbial community revealed that of the measured environmental variables, total dissolved solids was the only the significant correlation (p<0.05).
While cyanobacteria were abundant throughout all sites constituting between 22% and 52%, communities varied between regions of the Gulf.Outer bay sites closest to the inlet to Lake Victoria, Mbita East, and Naya, had high abundances (34% and 38% respectively) of chloroplast sequences matching Aulacoseira granulata var.angustissima with 100% identity, while sites in the Homa Bay region (Oluch to Mirunda Bay) had higher Nostocales abundances (22%-33%) and Inner Bay sites (Dunga to Bala Rawi) had higher Synechococcales abundances (Figure 3).Despite the variation in photoautotrophic group relative abundances, the presence and proportions of orders within other phyla were more consistent across sites.Bacteroidetes orders Chitinophagales, Cytophagales, Flavobacteriales, and Sphingobacterales, often associated with freshwater organic matter degradation, were moderately abundant at all sites.At the sites Mbita East and Naya, the order Rickettsiales was substantially higher abundances of 17% and 23% respectively compared to other sites.Closer examination revealed that the sequences represented a putative obligate endoparasite, with 100% sequence similarity to Candidatus Megaira polyxenophila that infects freshwater Paramecium.

Cyanobacterial assemblage distribution
Focusing on the cyanobacterial component of the communities, we found that Cyanobium spp.and Dolichospermum spp.were the dominant genera (Figure 4).We observed communities dominated by Dolichospermum spp. in the southernmost region of the western bay (sites 8-11; Figure 1) and northwestern region of the eastern bay (sites 14, 15, 17; Water temperature ( C), dissolved oxygen (mg L À1 ), and conductivity (μS cm À2 ).

Microscopy of cyanobacteria and eukaryotic phytoplankton
Through microscopy, we saw a similar distribution of cyanobacterial and eukaryotic phytoplankton as compared to the sequencing results from cruise A (Figure 5A, B).Analysis found that picocyanobacteria (Aphanocapsa, Aphanothece, and Chroococcus) were the primary cyanobacterial genera and Chlorophyta (green algae) were the dominant eukaryotic phytoplankton in the northeastern sampling sites.In the Homa Bay region, Dolichospermum were the prominent cyanobacteria and the eukaryotic phytoplankton distribution was distinct from other sites.At all sites other than Homa Bay (site 9), Bacillariophyta (diatoms) were present in abundance over 5%.Samples analysed from both cruise A and B (Nyando R. mouth, 3; Homa Bay, 9) showed minor differences in microscopy for the eukaryotic phytoplankton.At the Nyando R. mouth, cruise B samples contained Cryptomonas and microflagellates, while these taxa were absent in cruise A samples.Similarly, the cruise A sample from Homa Bay did not contain microflagellates.

Cyanotoxin gene detections
By analysing the cyanotoxin gene copies, we observed that both cyrA and mcyE/ndaF copies were found at nearly every site (Figure 6), while sxtA genes were not detected in any samples.Across the entire cruise track, mcyE/ndaF detections ranged from 6.4 Â 10 3 (Nyando R. mouth) to 1.2 Â 10 6 (Oluch) copies L À1 and cyrA detections ranged from 9.2 Â 10 3 (Mirunda Bay) to 1.2 Â 10 7 (Nyando R. mouth) copies L À1 .No mcyE/ ndaF copies were detected at Kendu Bay and no cyrA copies were detected at Homa Bay.In 11 of the 18 sites analysed, cyrA copies were found in higher abundance than mcyE/ndaF.At the sampling sites Oluch, Homa Bay, and Soklo, mcyE/ndaF copy concentrations were higher in comparison to all other sites.At Nyando R. mouth, cyrA gene copies were significantly higher than any other site, with 1.2 Â 10 7 copies L À1 detected (p = 0.02).

Eukaryotic community distribution
By examining the eukaryotic communities within the Winam Gulf, we were able to characterize and determine the distribution of major groups of photosynthetic eukaryotes (Figure 7).Distributions of eukaryotic divisions varied widely across sites with some discernable patterns.For example, dinoflagellates were in high abundance at many sites in the southern portion of the outer bay and near Homa Bay, with their abundance highest at Homa Bay (78%).Dinoflagellate communities were primarily comprised of Ceratium.Additionally, we observed that when dinoflagellate abundances were higher, Ochrophyta (belonging to the stramenopile group, inclusive of diatoms) abundances were generally lower and vice versa (r s = À0.88,p<0.05).Chlorophyta and stramenopiles were also found at all sites in greater than 1% abundance, except Homa Bay, with average abundances across the Gulf of 10% ± 5%

ENVIRONMENTAL MICROBIOLOGY REPORTS
F I G U R E 3 Relative abundance of prokaryotic orders.Orders below 1% abundance were aggregated at the phylum level.
F I G U R E 4 Relative abundance of cyanobacteria at the genus-level.
Although in relatively low overall abundance (2.67% ± 2.42%), fungi were present in greater than 1% abundance at many sites, except Homa Bay, Mirunda Bay, and Asembo Bay.Relative abundance was highest at the Nyando R. mouth (10%) and lowest at Asembo Bay (0.03%).In a few cases, sites with higher abundances of stramenopiles had higher abundances of fungi (r s = 0.59, p<0.05).At Dunga, relative fungi abundance was 6% and stramenopiles was 22%, and at Nyando R. mouth abundances were 10% and 28% for fungi and stramenopiles, respectively.

Clustering of sites through community similarities
Given the observed spatial patterns in prokaryotic and eukaryotic communities, we sought to identify distinct geographical clusters based on similarities in community compositions between sites.Using hierarchical sites was the pair Naya and Mbita East (0.31).Interestingly, the location within the Gulf appears to have a greater influence on community similarities than landscape features or hydrological characteristics, such as proximity to river mouths, as these sites did not cluster together.In contrast to the four well-defined groups, Mirunda Bay was clustered alone.

DISCUSSION
In this initial study of the Winam Gulf, we generated a dataset inclusive of all bacterioplankton with a focus on cyanobacteria and characterization of photosynthetic eukaryotic communities.Additionally, we were interested in the spatial distribution of cyanotoxins and toxigenic cyanobacteria.For both prokaryotic and eukaryotic communities, differences were noted that were aligned with their location in the Gulf, suggesting that localized physicochemical and/or unmeasured limnological factors were important in structuring the communities.Within the cyanobacteria, taxa associated with N-fixation were predominant, whereas Microcystis was only a minor part of the community.Microcystins were not frequently detected; however, toxigenic cyanobacteria were present at every site.

Toxigenic cyanobacteria were present, but not dominant
Prior to our study, Cyanobium spp.abundance in the Winam Gulf was not well described in the literature.
Cyanobium spp., part of the Synechococcus collective, were the dominant cyanobacteria at most sites (Salazar et al., 2020).At a few sites, Cyanobium spp.and Dolichospermum spp.were found at similar relative abundances.These cyanobacteria have co-occurred in previous systems since Cyanobium spp. is efficient at N acquisition and Dolichospermum spp.can fix N (Li et al., 2020).In Lake Erie, Cyanobium and Synechococcus OTUs are described in 16S rRNA gene sequencing data, but microscopy often does not allow for resolution beyond picocyanobacteria (Berry et al., 2017;Matson et al., 2020;Ouellette et al., 2006).We saw that through microscopy, the genera of picocyanobacteria identified differed from sequenced samples where ASVs were assigned to Cyanobium.Identification of picocyanobacteria can be difficult due to size, but databases for assigning taxonomy may also be limited and there is no complete consensus between morphology and 16S rRNA gene sequences (Jasser & Callieri, 2016;Yarza et al., 2014).The variability in taxonomic assignments from these samples emphasizes the importance of both traditional microscopy and sequencing approaches.
Microcystis spp.has been traditionally recognized as a concern in the Winam Gulf, however, at time of sampling it was not a prominent part of the community (Sitoki et al., 2012).The low abundance of Microcystis spp.may be attributed to recent changes in water flow due to the removal of the Mbita causeway blocking flow from the open waters of Lake Victoria (Simiyu et al., 2022).Replacement of the causeway with a bridge has lowered conductivity and has been linked to increased diatom abundance and decreased Microcystis populations (Simiyu et al., 2022).At most, during our study, Microcystis spp. was $7% of the community at Soklo.In microscopy samples from cruise B, we observed a higher relative abundance at the Sondu R. mouth as compared to what was found through sequencing from cruise A. Despite the short amount of time between cruises, the Microcystis spp.community increased in density due to growth and/or movement of the bloom, but due to lack of continuous satellite imagery, we are unable to determine the exact factor contributing to the increase in abundance.There was an apparent association between Microcystis spp.and Pseudanabaena spp.presence (r s = 0.45, p = 0.07).The small, cosmopolitan cyanobacteria Pseudanabaena spp. is often associated with Microcystis spp.colonies (Agha et al., 2016;Smith et al., 2021).Pseudanabaena spp.typically grows within the mucilage of Microcystis spp.colonies and this association is likely due to regulation of light levels and access to nutrients (Agha et al., 2016).
Though the bloom was not actively producing microcystins when cruise A samples were collected, it was found to be toxigenic, with mcyE/ndaF detected at all sites except Kendu Bay.Therefore, the cyanobacteria bearing these toxin genes could initiate microcystin production under favourable conditions (Beversdorf et al., 2015).In contrast, cruise B found elevated microcystin concentrations in the Homa Bay region, but Microcystis spp. was not identified in either cruise microscopy samples.As Microcystis spp. was in low relative abundance in the sequencing data, there is potential that this small community was not captured by microscopy.The World Health Organization (WHO) has guidelines for exposure to cHAB blooms based on chlorophyll-a levels with increased risk of health effects above 50 μg L À1 (Codd et al., 2005).Chlorophyll-a levels at many sites in the Winam Gulf greatly exceeded WHO thresholds, yet microcystin concentrations were primarily below detection, with the exception of the Homa Bay Pier, which was slightly higher than WHO guidelines of 1.0 μg L À1 for microcystins in drinking water.
We suspect that Microcystis spp.were responsible for the majority of the mcyE/ndaF gene detections, as they generally followed trends in Microcystis spp.abundance rather than Dolichospermum spp.Moreover, we isolated a toxigenic Microcystis panniformis from water sampled near Homa Bay, further supporting that Microcystis spp.may be contributors of microcystin synthetase gene copies (Brown et al., 2024).

First detection of the cylindrospermopsin synthetase gene in the Winam Gulf
Although identified previously in the Gulf, Cylindrospermopsis spp. was not recognized as a cyanobacterial genus of high concern (Sitoki et al., 2012).In this study, Cylindrospermopsis spp. was detected in all sites, but in about half of the sites, abundance was extremely low within the community.At the site with the highest abundance of Cylindrospermopsis spp., the Nyando R. mouth, there was no visible surface bloom and there was no association between higher turbidity and abundance of Cylindrospermopsis spp.(r s = À0.004,p = 0.9894).At the river mouth, sample water was turbid and had a large amount of visible sediment.However, Cylindrospermopsis spp.blooms can be facilitated by increases in turbidity, as it is adapted to a broad range of light conditions (Burford et al., 2016;Wood et al., 2014).The 16S detections of Cylindrospermopsis spp. at Nyando R. mouth were confirmed through microscopy, where 6300 cells mL À1 were counted.
Detection of the cyrA gene at nearly all sampling sites was unexpected.Cylindrospermopsins have been measured in open Lake Victoria in minimal concentrations (0.004-0.01 μg L À1 ), far below WHO drinking water guidelines, but we have not encountered any reports of this cyanotoxin in the Winam Gulf (Geneva: World Health Organization, 2020; Mchau et al., 2021;Omara et al., 2023).There is some potential that other cyanobacteria detected (Cuspidothrix spp.and/or Dolichospermum spp.) make up part of the cyrA-containing population but given the covarying trends in Cylindrospermopsis spp.abundances and cyrA copy concentrations, it is likely that Cylindrospermopsis is primarily responsible (Geneva: World Health Organization, 2020).This is best illustrated at the Nyando R. mouth, where relative abundance and cyrA copies were both highest (6% and 1.2 Â 10 7 , respectively).Previously, it was found that C. raciborskii AWT205 contained 1.13 ± 0.19 copies of cyrA per cell (Al-Tebrineh et al., 2012).In our study, with 1.2 Â 10 7 cyrA copies L À1 and a population of 6.3 Â 10 6 cells L À1 , each cell would have about two copies of cyrA, assuming all Cylindrospermopsis spp.cells were toxigenic.This suggests that the dominant Winam Gulf strains have more cyrA copies per cell than AWT205, so Cuspidothrix and Dolichospermum may also be contributing to the total.
Both Cylindrospermopsis spp.and cylindrospermopsins (therefore cyrA) are common in tropical regions.While Cylindrospermopsis spp.and cyrA genes have been detected in a number of African surface waters, it appears that distribution of cylindrospermopsins themselves is understudied (Kokoci nski et al., 2017;Moreira et al., 2021;Scarlett et al., 2020).These novel detections provide evidence of potential cylindrospermopsin production in the Winam Gulf and can help to improve future cyanotoxin detection efforts.This also provides support for informing the public that lake water appearing to have no bloom may contain cyanobacteria and toxins.However, despite there being regulations for cylindrospermopsins and other cyanotoxins in drinking water, many communities surrounding the Gulf and in the global south as a whole do not have consistent access to processed drinking water.One previous survey found that about 50% of the surveyed group from Winam Gulf communities had no options other than using raw lake water (Roegner et al., 2020).Therefore, these guidelines in this region may often be of little importance to many people and are overall more useful to communities that have consistent and widespread availability of treated drinking water.

Spatial distribution of prokaryotic and eukaryotic communities
Over both sampling periods, the physicochemical parameters were consistent throughout the Winam Gulf, with the exception of DO.The fluctuations in DO concentrations could have been a result of varying rates of photosynthesis at different times of day when sampling occurred.Sites with high and low conductivity correspond to two notable locations, the Nyando R. mouth and the Rusinga Channel.As the Nyando River flows through fertilized farmland and tea plantations, accumulating solutes from runoff likely contribute to higher conductivity waters entering the Gulf (Raburu & Okeyo-Owuor, 2006;Gabellone et al., 2005).Conversely, the conductivity of open Lake Victoria is low, and through mixing yielded the lower conductivity observed at Mbita East, as previously found in the Winam Gulf (Simiyu et al., 2022;Sitoki et al., 2010).Overall, with the exception of these locations, this indicates that the Gulf is well-mixed and only few distinct water masses are present, where water chemistry differences might be a key determinant of microbial community composition.
To view how diversity and abundances were related among sites, we used Bray-Curtis dissimilarity to cluster sites together based on the entire community.For prokaryotic communities, we observed that in the sites surrounding Homa Bay (Cluster 1), the order Nostocales was in relatively high abundance (22%-33%).Asembo Bay was in Cluster 1 as well because of a Dolichospermum spp.dominated community.Similarly, sites with greater than 1% Cylindrospermopsis spp.abundance and high cyrA copy concentrations were also clustered together (Cluster 4).Due to the similarities of other bacterial phyla at the sampling sites, the Bray-Curtis clustering most closely follows trends in cyanobacterial genera.
While cyanobacteria were responsible for the Winam Gulf bloom, eukaryotic phytoplankton were major contributors.Several photosynthetic eukaryotes were found by analysing the 18S rRNA, including dinoflagellates and diatoms (Ochrophyta).We observed similar trends of high dinoflagellate abundance at Kisumu Pier and Homa Bay, but not at Sondu R. mouth (Kundu et al., 2017;Lung'Ayia et al., 2000).Diatoms have been historically found in Lake Victoria and the Winam Gulf, but as stated prior, diatom communities have shifted as a result of eutrophication (Njagi et al., 2022).However, the two sites near the Rusinga Channel had higher abundances of diatoms and this is likely a direct result of water exchange from the open lake and is indicative of less eutrophic waters (Simiyu & Kurmayer, 2022).This is further supported by the high abundance of chloroplast reads in these two sites that classified to Bacillariophyta.While less abundant overall, Chlorophyta and Cryptophyta were always present and sometimes exceeded relative abundances of the other eukaryotic phytoplankton.
Along with eukaryotic phytoplankton, we were interested in finding potential parasites that may be correlated with these communities.Within the true fungi community are many taxa known to be parasitic, and while these communities are of low abundance it is worth pointing out that several of these species are known to infect diatoms.Many of the most abundant fungi that are parasitic belong to the phylum of Chytridiomycota (chytrids).Within the chytrids are the genera Zygophlyctis (formerly Zygorhizidium), and these genera are known to parasitize diatoms Aulacoseira and Ulnaria (formerly Synedra), both of which were found within the Gulf (Seto et al., 2020).Mbita East, Naya, and Nyando R. mouth had the highest relative abundances of diatoms but only the Nyando R. mouth had high abundances of fungi.Since Mbita East and Naya are located in the channel, it is likely that these waters are turbulent from seiche activity.Consequently, such water movement could be limiting chytrid pathogenesis, as described by McKindles et al. (2021) and Wagner et al. (2023).

CONCLUSIONS
Above we described characterization of bacterial and eukaryotic communities of the Winam Gulf, Kenya, and the first detections of cylindrospermopsin synthetase genes (cyrA) indicating potential for cylindrospermopsin production.We primarily found that bacterial communities at a high level were similar among all sites and that the cyanobacterial communities were diverse having different dominant genera between sites.As microcystins have been the most highlighted cyanotoxin of concern in the Gulf, finding potential for cylindrospermopsin production emphasizes the need for broader cyanotoxin monitoring (Plisnier et al., 2022).
Many people using lake water are aware that green water or a surface scum may be harmful to use and contain cyanotoxins, but at the site with highest cyrA copy concentration (Nyando R. mouth), no bloom was visibly apparent (Obuya et al., 2023;Secaira Ziegler et al., 2022).The risk of cyanotoxin consumption is high in these cases where the cyanobacteria may be distributed throughout the water column and obscured by suspended sediment.The triggers for cyanotoxin production are also not well-characterized, with differential effects from temperature, nutrients, and light that are often different between cyanobacterial genera (Chaffin et al., 2018;Dyble et al., 2006;Mesquita et al., 2019;Paerl & Otten, 2013;Wagner et al., 2021).Additionally, the triggers for cyanotoxin production in the Gulf and other African waterbodies may differ from North American and European waterbodies, where many of these studies are done.
We suggest further research into cyanotoxin production in the Gulf, from the most abundant cyanobacteria present.We also suggest continual monitoring of bacterial communities to describe how communities change over the year, and between years.Ongoing monitoring will also result in stronger environmental datasets for analysis alongside community data.Continuing studies are warranted to determine how communities and cyanobacterial dominance changes between the rainy and dry seasons, potentially as a result of changing nutrient runoff.Most important of all, regular monitoring of cyanotoxins in surface waters and drinking water intakes could reduce exposures to those using raw lake water.
Map of both cruise tracks.Sondu R. mouth and Nyando R. mouth are sites near the river mouth for the Sondu and Nyando respectively.(B) 2022 Cruise tracks A and B overlaid on a satellite image from the Gulf (taken 30 June 2022).

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I G U R E 5 Microscopy counts of (A) cyanobacterial genera and (B) eukaryotic phytoplankton.Taxa below 5% abundance were aggregated at the phylum level.BACTERIAL COMMUNITY AND CYANOTOXIN GENE DISTRIBUTION 9 of 17 ENVIRONMENTAL MICROBIOLOGY REPORTS clustering of prokaryotic communities, we found that sites clustered into three distinct branches, with 5 total clusters, arranged mostly in line with the sites' geographical location within the Gulf.The three branches generally represented the sites near the Rusinga Channel (Naya, Mbita East; Cluster 5), within/near Homa Bay (Homa Bay, Oluch, Soklo, Asembo Bay; Cluster 1), Mirunda Bay (Cluster 2), and the eastern Gulf, including the remaining sites grouped together (Figure 8).The exception to the geographical clustering trend was Asembo Bay, which was found in Cluster 1 with Homa Bay.The pairs Homa Bay and Oluch were most similar to one another (Bray-Curtis dissimilarity = 0.34) as well as Gingra and Bala Rawi (0.29).Dunga and Maboko Island were most related in another cluster (0.33), and most distant from the other F I G U R E 6 Cyanotoxin synthetase gene (mcyE/ndaF, cyrA) detections in the Winam Gulf sampling sites during the 2022 cruise.Y-axis is on a log scale.Error bars represent standard deviation of n = 3. F I G U R E 7 Relative abundance of eukaryotes at the division level.

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I G U R E 8 Hierarchical clustering of sampling sites based on compositional similarities of prokaryotic communities.
Chlorophyll-a concentrations of the Winam Gulf sampling sites.All sites other than Soklo and Asembo Bay include data from both cruises, n = 3.
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